EIA test using nondenatured HIV antigen for early detection of HIV infection

ABSTRACT

Recombinant human immunodeficiency virus antigens capable of immunologically identifying the presence of early anti-HIV antibodies are stably expressed in a number of cell lines. These antigens have several clinically important applications as non-hazardous tools in the detection of human immunodeficiency virus exposure/infection, and in screening methods for HIV infection in idiopathic chronic lymphopenia (ICL). These techniques are improved over existing immunologically based and PCR based detection methods, as they provide for the detection of infection/exposure in samples determined to be negative by conventional forms of these types of assays that do not detect anti-HIV gp16O antibodies that react to conformational epitopes of HIV. The invention finds particular application in the detection of human immunodeficiency virus exposure/infection in infants. The earlier detection of the described methods is provided through the preserved immunoreactivity of the described recombinant conformationally intact human immunodeficiency virus that is capable of detecting a class of “early” anti-human immunodeficiency virus antibody not previously detectable by standard Western Blot or ELISA methods. The human immunodeficiency virus gp16O envelope antigen comprises one of the specific recombinant antigens examined with clinical human samples in these improved screening methods.

[0001] The government owns rights in the present invention pursuant togrant number ROI-AI32444 from the National Institutes of Health.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the fields ofdiagnostic and screening tests. More particularly, it concerns improvedmethods for the detection of early anti-HIV antibody in a sample, aswell as early diagnostic tests to detect human immunodeficiency virusexposure or infection in infants. An additional aspect of the inventionconcerns methods for detecting HIV in idiopathic chronic lymphopeniapatients. The invention also relates to the field of recombinantproteins, as particular recombinant proteins and recombinant proteincomposite preparations of several human immunodeficiency virus strainsare disclosed. The invention also relates to the field of commercialdiagnostic and prognostic assay plates and kits, as assay platesdesigned to detect an early anti-HIV antibody that include the describedrecombinant human immunodeficiency virus antigen or preparations ofhuman immunodeficiency virus infected cell isolates are described.

[0004] II. Background of the Invention

[0005] Patients infected with human immunodeficiency virus (HIV) areknown to eventually mount a humoral immune response to the virus. Theproduction of anti-HIV antibody is one marker used to detect thisresponse. In some studies, anti-HIV antibody has been reported to bemore reliable for diagnosis than either HIV culture or HIV antigendetection in patient samples ^(1,2). Consequently, anti-HIV antibodydetection tests are the most common method of diagnosis of infection.Both EIA and Western blot assays are currently used in the detection ofanti-HIV antibody.

[0006] Unfortunately, a degree of unreliability continues to exist withthe use of conventional anti-HIV antibody screening methods, such as byconventional EIAs. Thus, a further confirmatory test, such as a WesternBlot (WB) or fixed-cell immunofluorescence assay, have becomerecommended additional testing procedures.

[0007] Despite these and other additional precautionary testingmeasures, a number of studies report the existence of a seemingly silentperiod of HIV infection during which antibody to the virus is notdetectable even after exhaustive testing. This period reportedly extendsfrom the point of infection to the time infection is detectable throughconventional sero-conversion assays. This silent period has beenreported to persist anywhere from a few months to as much as two andone-half years before infection is detectable by conventional EIAs andWestern blot assays.

[0008] While not always successful, culturing of peripheral bloodlymphocytes to amplify HIV does provide for detection of the virus whenanti-HIV antibody cannot be detected by conventional EIA or WB. However,several recent studies using PCR-based HIV detection methods continue toreport the existence of PCR(+)positive, sero(−)negative cases inhigh-risk populations¹⁰⁻¹⁷. Nevertheless, PCR usually does detectinfection before conventional sero-conversion methods, with theaforedescribed period of silent infection being reduced by approximatelyone month, at least in some cases¹⁸.

[0009] The rate of HIV transmission in negatively tested blood, usingconventional testing methods, continues to persist at a relativelyconstant rate²⁵. For example, HIV-1 transmission from seemingly“seronegative” blood using EIA conventional methods, continue to bereported²¹⁻²³. Donated organs also constitute a source of HIV diseasetransmission, with HIV infection being diagnosed in recipients of organsfrom individuals whom, again, test HIV seronegative by conventionalassays²⁴.

[0010] Retrospective studies have reported that early donor educationand self-exclusion measures has reduced the rate of diseasetransmission²⁶. However, such exclusion methods together with antibodytesting, while hopefully reducing the probability of at least some falsenegative results²⁷, provides only a partial and imperfect solution tothe problem in at least a small subset of reported HIV cases.

[0011] Some studies report the presence of HIV specific T-cells in highrisk individuals testing negative with conventional EIA, WB, and PCRbased detection techniques^(28,29). Other reports have identified theexistence of B-cells which produce HIV-specific antibodies in vitro thatare present in EIA-negative, WB-negative, high-risk subjects³⁰. Whilethese approaches present possible alternatives, for testing, they arerelatively complex and difficult procedures, and are thus impracticalfor large-scale clinical screening. The expense and time associated withthis type of testing again leaves a need in the medical arts for areliable and practical HIV screening and detection approach.

[0012] Early HIV infection of infants is a particularly troublesomeproblem. Current technology renders it difficult to diagnose whether aninfant less than 18 months of age is infected, absent development ofovert clinical symptoms. Conventional HIV serological tests for anti-HIVantibody are inadequate for detecting infection in an infant because theantibody detected is not necessarily that of the infant, but is that ofthe HIV-positive mother. This maternally derived antibody typicallypersists for up to 21 months in the infants system³⁴.

[0013] Neither IgA or IgM antibody cross the placenta. Hence, studies inchildren have emphasized the detection of IgA and IgM as indicators ofinfant HIV infection. In one study, both HIV-specific IgA and IgM werefound in infants up to 12 months of age born to sero-positive mothers,with twice as many samples yielding IgA anti-HIV compared to IgM (66%vs. 33%)³⁵ using conventional screening assays (WB, EIA).

[0014] Currently, approximately 50% of infected infants can beidentified at birth, approximately 90% by 3 months of age, and almostall by 6 months of age using combination HIV culture, PCR, IgA antibodytests, and p24 antigen tests³⁸. However, the fact that HIV can bedetected in only one-half of infected infants at the time of birth againpoints to the continued need for improved early HIV detection ininfants.

SUMMARY OF THE INVENTION

[0015] The present invention, in a general and overall sense, concernsrecombinant HIV envelope proteins and peptides, and early anti-HIVantibody immunoreactive fragments thereof, that are capable ofimmunologically binding to early anti-HIV antibodies.

[0016] As used in the description of the present invention, earlyanti-HIV antibodies are defined as the first anti-human immunodeficiencyvirus antibodies that are induced in a human infected with the HIVvirus, these antibodies being capable of recognizing conformationalepitopes of HIV gp160 antigen and which are not detectable by currentEIA or Western Blot assay using HIV gp160 target antigen that has nothave retained conformational epitopes.

[0017] The invention further provides for an improved HIV detection andscreening method by allowing for the identification of early anti-HIVantibody. These early anti-HIV antibodies previously went undetectedusing conventional assays because the target antigens historicallyemployed in these assays lacked sufficiently preserved conformationalepitopes necessary for early antibody recognition.

[0018] The compositions and assays of the present invention compriseimproved HIV target antigens that include the conformational epitopes ofan HIV envelope protein, specifically the HIV gp160. The early anti-HIVantibodies detectable using the described antigen do not recognizeprimary sequence epitopes, and hence they go undetected withconventional serological tests that employ at least partially denaturedHIV target antigen (EIA and Western blot).

[0019] Because PCR-based assays provide little improvement overconventional EIA and Western Blot serological-based assays for detectingHIV, it is expected that the presently described EIA's will also providean improved method over PCR based detection and screening methods.

RECOMBINANT PROTEINS AND PEPTIDES OF HUMAN IMMUNODEFICIENCY VIRUS

[0020] In some embodiments of the invention, a recombinant proteincomprising a recombinant human immunodeficiency virus envelope proteincapable of immunologically binding an early anti-HIV antibody isprovided. For example, these recombinant proteins in particularembodiments may be further defined as having a molecular weight of about160,000 Daltons as determined by SDS/PAGE. In these particular forms,the recombinant HIV protein is an HIV envelope protein, HIV gp160.

[0021] The recombinant HIV envelope proteins of the invention mayfurther be described by reference to the process by which they areprepared. For example, in one embodiment, the process comprisespreparing a nucleic acid sequence or a restriction fragment of the humanimmunodeficiency virus nucleic acid sequence that encodes a humanimmunodeficiency virus envelope protein, such as gp160; inserting therestriction fragment into a vector capable of transfecting a eukaryoticcell to provide a recombinant vector; transfecting eukaryotic cellcapable of expressing the human immunodeficiency virus envelope proteincapable of binding an early anti-HIV antibody to provide a recombinanteukaryotic cell; culturing the recombinant mammalian cell underconditions suitable for expression of the recombinant HIV protein; andcollecting recombinant HIV envelope protein capable of binding earlyanti-HIV antibody.

[0022] An example of a mammalian cell capable of expressing the HIVenvelope protein nucleic acid sequence as described here is the CEM cellline. Such CEM cell lines are available through the American TypeCulture Collection (ATCC). An example of the vector capable oftransfecting a mammalian cell is a retroviral vector, such as pLNSX.Other carriers, such as an adenovirus or a plasmid, may also be used totransfer the HIV envelope protein encoding nucleic acid sequence. Therecombinant retroviral vector in a particular embodiment of the processis the pLNSX-env.

[0023] In other embodiments of the recombinant human immunodeficiencyvirus protein, the protein is described as comprising a recombinantgp160 human immunodeficiency virus glycoprotein or fragments of thegp160 protein, such as the gp120 or gp41 fragment of the gp160 protein.

[0024] The recombinant HIV envelope proteins and peptides of theinvention are further described in some embodiments as comprisingrecombinant proteins and peptides prepared from substantially purifiedand non-denatured lysates of mammalian cells transfected with vectorsexpressing recombinant human immunodeficiency virus, as well asrecombinant proteins/peptides comprising the substantially purified andnon-denatured expression products of these transfected mammalian cells.

[0025] In yet other embodiments, the recombinant protein/peptidepreparations comprise a composite of recombinant human immunodeficiencyvirus gp160 proteins, the composite comprising recombinant gp160 proteinfrom more than one human immunodeficiency virus strain.

[0026] Mammalian cell lines that are transfected with retrovirusexpression vectors carrying the gp160 encoding-human immunodeficiencyvirus gene have been prepared with gp160-encoding nucleic acid fragmentsobtained from several different strains of the human immunodeficiencyvirus obtained from HIV-infected patients. For example, recombinant HIVenvelope protein has been prepared from HIV₂₁₃ virus strain.

[0027] In a particular embodiment, the protein/peptide gp160 compositepreparation comprises the recombinant gp160 protein expression productof at least three different HIV strains found to be immunoreactive withthe early HIV antibody detected in a representative number of serumsamples determined to be seronegative by conventional EIA and WesternBlot techniques. By way of example, three such HIV strains are HIV₂₁₃,HIV_(C) and HIV_(AC-1). These HIV strains have been deposited with theAmerican Type Culture Collection depository (12301 Park Lawn Drive,Rockville, Md. 20852). The deposit information is as follows: HIV₂₁₃ -ATCC VR 2247 HIV_(C) - ATCC VR      HIV_(AC-1) - ATCC VR 2246HIV_(TP-1) - ATCC VR 2245

[0028] Target antigen may be prepared from either the expression productobtained from eukaryotic cells transfected with retroviral or othervectors carrying these gp160 encoding nucleic acid sequences orfragments thereof, or from substantially nondenatured lysates of suchtransfected eukaryotic cells.

[0029] As used in the description of the present invention in thedescription of the human immunodeficiency virus recombinantprotein/peptide preparations and cell lysates, the phrase “substantiallynon-denatured” in used to define a protein having a preservedconfigurational integrity of the human immunodeficiency virus envelopegp160 protein or a portion thereof sufficient to bind early anti-HIVantibody. Conformation of the protein/peptide used as target antigen isimportant in providing this early antibody recognition.

[0030] The present inventor provides here procedures that preservesufficient conformational integrity of the protein/peptide to allowearly anti-HIV binding recognition. It is anticipated that given thedisclosure here, other similar protein/peptide preparation processes maybe devised that result in useful target antigen compositions for earlyanti-human immunodeficiency virus screening. All such modifiedprocedures, insofar as they represent minor or insignificantmodification of the procedures and specific materials described herein,are therefore intended by the inventor to be embraced within the scopeof the present invention.

[0031] While a number of different HIV strains were examined by thepresent inventor, other HIV viral strains not specifically mentioned orexamined here may also be employed in the preparation of the various HIVproteins/peptides of the invention. It is expected that other HIV viralstrains may be used to provide the defined substantially preservedconformational epitopically intact HIV proteins capable of earlyanti-HIV antibody recognition. The particularly noted HIV strains usedto create recombinant protein/peptide target antigen were selected basedon an observed activity to bind early anti-HIV antibody in human patientserum or plasma samples determined to be seronegative by conventionalantibody testing procedures. Hence, additional such representativestrains may be identified and selected using the procedures outlinedherein, and subsequently processed again according to the proceduresdescribed in detail here in providing recombinant HIV antigen alsouseful in screening and diagnosing early anti-HIV antibody and HIVinfection in a patient sample.

[0032] In a particular embodiment, the recombinant protein/peptide ofthe invention comprises a sufficiently conformationally intact HIVenvelope protein capable of immunologically binding an early anti-HIVantibody, said antigen being isolatable as an expression product from amammalian or other eukaryotic cell transfected with an expression vectorhaving a sequence encoding a gp160 HIV envelope protein. The HIV gp160envelope protein, by way of example, may be that expressed by aeukaryotic cell infected with a viral strain HIV_(C), HIV₂₁₃, orHIV_(AC-1). It is also expected that other HIV isolate strains that arecapable of expressing the gp160 antigen, or instead the gp41 or gp120HIV envelope antigen, may be used in conjunction with the presentinvention as well.

PURIFIED PREPARATIONS OF HIV ENVELOPE PROTEINS

[0033] Further aspects of the present invention concern thepurification, and in particular embodiments, the substantialpurification, of the described recombinant HIV envelope proteinpreparations. The term “purified envelope protein” as used herein, isintended to refer to a protein composition that is isolatable fromeukaryotic cells, either in the form of a solubilized population of HIVinfected cells or as a protein from recombinant-expressing cells. Thesepurified envelope proteins are further defined as essentiallynon-denatured and capable of binding with early anti-HIV antibody. TheHIV envelope protein is purified to any degree relative to itsnaturally-obtainable state. In some embodiments, the purified antigenstate is relative to purity of the recombinant antigen as it exists in awhole mammalian cell lysate. A purified HIV envelope protein thereforealso refers to a recombinant envelope protein, free from the environmentin which it may naturally occur in intact recombinant-expressingmammalian cells.

[0034] Generally, “purified” will refer to an HIV envelope proteincomposition wherein various non-HIV envelope components, such as othercell components, have been removed, and which composition substantiallyretains its antigenicity and/or capacity to interact with early anti-HIVantibody. Where the term “substantially purified” is used, this willrefer to a composition in which the human immunodeficiency virusenvelope protein forms the major component of the composition, such asconstituting about 50%, about 60%, about 80% or about 95% of the proteinin the composition or more.

[0035] Various methods for quantifying the degree of purification of theHIV protein will be known to those of skill in the art in light of thepresent disclosure. These include, for example, determining the activityof the preparation for detecting early anti-HIV antibody by assessingthe number of different polypeptides within a fraction by SDS/PAGEanalysis. In some embodiments, the recombinant HIV protein is to bepurified to homogeneity expressed as an about 80% purity from a crudecell lysate so as to be identifiable in a single band of an SDS page gelstained with comassie blue. SDS page gel analysis of a proteinpreparation is well known to those of ordinary skill in the art.

[0036] Various techniques suitable for use in protein purification willbe well known to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. A specific example presentedherein is the purification of recombinant gp/60 expressed mammaliancells which are solubilized using affinity binding to anti-gp41monoclonal antibody bound to wells of plastic EIA plates.

[0037] Some of the recombinant HIV protein preparations from transfectedmammalian cell lysate of the present invention have a purity of 80%.This number represents a ratio of, for example, 8 gp160 HIV antigenmolecules to two non-gp160 molecules. Preparations of lower or higherpurity are also useful in detecting the early anti-HIV antibody.

[0038] The preferred purification method disclosed hereinbelow containsseveral steps and represents the best mode presently known by theinventors to prepare a substantially purified recombinant HIV envelopeprotein and still maintain its native conformation. This method iscurrently preferred as it results in the substantial purification of therecombinant HIV protein, as assessed by SDS-PAGE determinations andability to bind to early antibodies, while simultaneously coating EIAplates. This preferred mode of recombinant protein purification from HIVtransfected mammalian cells involves the execution of certain steps inthe order described hereinbelow. However, as is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified recombinant HIV envelope protein, such as therecombinant gp160 HIV envelope protein.

[0039] As mentioned above, although preferred for use in certainembodiments, there is no general requirement that the HIV transfectedmammalian cell lysate always be provided in their most purified state.Indeed, it is contemplated that less substantially purified celllysates, which are nonetheless enriched in recombinant HIV envelopeprotein, such as gp160 relative to the natural state, will have utilityin certain embodiments. These include, for example, the detection ofearly anti-HIV antibody. Partially purified HIV transfected mammaliancell lysate fractions for use in such embodiments may be obtained bysubjecting an HIV transfected mammalian cell lysate extract to one or acombination of the steps described herein, the HIV recombinant envelopepreparations may also be obtained as a secreted product from cellscarrying an HIV envelope antigen (e.g. gp160) encoding nucleic acidsequence.

RECOMBINANT VECTORS

[0040] The recombinant vectors of the invention may comprise any vehiclecapable of transfecting a eukaryotic cell. By way of example, suchvehicles include retrovirus vectors and adenovirus vectors. Plasmids mayalso be used to transfer HIV nucleic acid fragments encoding the HIVenvelope proteins of the invention. In some embodiments, the retroviralvector comprises a nucleic acid sequence encoding a humanimmunodeficiency virus envelope protein, such as the gp160, or thatencodes an early anti-HIV antibody immunoreactive fragment of the humanimmunodeficiency virus envelope protein.

[0041] In particular embodiments, the nucleic acid sequence encoding thehuman immunodeficiency virus envelope protein comprises an SalI-XhoIrestriction fragment of a human immunodeficiency virus nucleic acidsequence. By way of example, the inventor has prepared SalI-XhoIrestriction fragments of several human immunodeficiency viruses,particularly HIV strains C, 213 and AC-1, and blunt-end ligated thefragment into a retroviral vector, such as the pLNSX retroviral vector.

MAMMALIAN CELL LINES

[0042] The present invention in another aspect provide recombinantmammalian cells that express the recombinant human immunodeficiencyvirus envelope proteins and peptides. Any variety of mammalian cells orcell lines may be used, as long as they are capable of expressing therecombinant HIV protein capable of immunologically detecting the earlyanti-HIV antibody. By way of example, a cell line that may be used is aCEM human cell line, particularly described as CEM human T-cells.

METHODS FOR PREPARING RECOMBINANT HIV ENVELOPE PROTEIN AND PEPTIDE

[0043] Methods for preparing the recombinant HIV envelope are alsoprovided. In one particular embodiment, the method comprisestransfecting a mammalian cell with a retroviral vector geneticallyengineered to include a sequence encoding a human immunodeficiency viralenvelope protein. These transfected mammalian cells are then subject toa screening process, such as by antibiotic resistance to G418 forexample, and clones selected that express the highest amount of thehuman immunodeficiency virus envelope protein. By way of example, clonesexpressing the highest amounts of HIV envelope protein could be selectedusing an anti-HIV gp160 monoclonal antibody (e.g., DZ33, Medarex, Inc.).

[0044] One particular clone expressing the HIV envelope protein gp160isolated by the present inventors is Clone 7. Clone 7 was obtained bytransfection of a CEM cell line with LNSX retroviral vector containingthe env gene of HIV₂₁₃ strain. This clone 7 was examined by flowcytometry, and shown to express high levels of the recombinant HIV gp160protein (FIG. 5).

[0045] Other clones that express a fusion protein of gp160 obtained frommore than one of the HIV strains may also be obtained. Such would beachieved, for example, by constructing a retroviral vector that includeda gp160 encoding fragment of nucleic acid obtained from the desired HIVisolates, and then transfecting a mammalian cell line with saidconstruct.

[0046] Any of the herein described recombinant HIV antigens may be usedalone or in combination as a target antigen for the detection of earlyanti-HIV antibody. Combinations of these recombinant antigens from thenoted viral strains have proven to be particularly efficacious fordetecting the early anti-HIV antibody in the widest range of patientsamples examined to date.

METHODS/PROCESSES OF PRODUCING RECOMBINANT HIV ENVELOPE PROTEIN/PEPTIDE

[0047] The invention also provides for methods/processes of producing arecombinant HIV antigen. In one embodiment, the method comprisesobtaining a nucleic acid fragment encoding a human immunodeficiencyvirus gp160 envelope protein; ligating the nucleic acid fragment into avector to provide a recombinant vector; transfecting a mammalian cellwith the recombinant vector to provide a transfected mammalian cell; andcollecting recombinant gp160 protein. The recombinant gp160 protein maybe collected by virtue of solubilizing the infected mammalian cell in asolubilizing agent that does not destroy the early anti-HIV antibodydetecting capability of the antigen, such as digitonin, and thenremoving the cellular components in the solubilized preparation.Alternatively, the recombinant HIV antigen may be collected as asecreted product from recombinant eukaryotic cells, such as from a yeastcell that has been genetically engineered such that it secretes thegp160 HIV envelope protein.

[0048] A method that was used to collect recombinant gp160 that was usedas target antigen to detect early anti-HIV antibody in a sample wasthrough the use of 1.0% digitonin as a solubilizing agent on CEM cellsinfected with HIV. This procedure is described at Example 3. Therecombinant HIV gp160 antigen was captured onto an ELISA plate bypouring the whole recombinant cell lysate into wells of the plate, whichwill be coated with mouse anti-gp41 muAB. The plate was then washed toremove cellular components and other antigens. In this fashion, thedesired HIV gp160 antigen was isolated on the plate.

[0049] In a particular embodiment, the method or preparing a recombinantgp160 envelope protein comprises: obtaining a nucleic acid fragmentencoding a gp160 HIV envelope protein; inserting said fragment into avector capable of transfecting a mammalian cell; and transfecting amammalian cell capable of expressing the gp160 envelope protein withsaid vector. The method may include the further step of collecting therecombinant gp160 protein.

ASSAY PLATES

[0050] In some embodiments, the wells of the assay plates may first becoated with an anti-gp41or anti-gp160 antibody. This would immobilizeHIV gp160 antigen to the plastic in the presence of a mild solubilizingbuffer, such as from about 0.1% to about 10% digitonin (particularlyabout 1% digitonin). Such an approach is particularly efficacious inpreparing assay plates with wells made of plastic.

[0051] The assay plates in other embodiments of the invention comprise amultiplicity of microtiter wells, and in some embodiments, polystyrenemicrotiter wells. These wells would be coated with about 500 ng/well ofthe recombinant HIV envelope protein, or recombinant HIV antigen orHIV-infected whole cells or cell lysates thereof.

EARLY ANTI-HIV ANTIBODY DETECTION ASSAYS

[0052] In yet another embodiment, the invention provides for improvedanti-HIV antibody detection assays using the aforedescribed native orrecombinant HIV proteins/peptides as target antigen. These improvedassays, particularly ELISA sandwich-type assays, provide for thedetection of early anti-HIV antibodies in sandwich-type assays, providefor detection of early anti-HIV antibody in samples that test anti-HIVantibody negative using conventional EIA and WB techniques.

[0053] The format of the EIA may be described as employing plates thatare directly coated with the HIV antigen (the antigen being eitherrecombinant HIV envelope protein expressed from HIV-transfectedmammalian cells, or a lysate of substantially non-denatured, solubilizedHIV-infected mammalian cells that express the HIV envelope protein), orplates that are designed to function as an antibody capture sandwichassay.

[0054] Immunoassays

[0055] As noted, it is proposed that the recombinant gp160 of theinvention will find utility as immunogens, e.g., in connection withvaccine development, or as antigens in immunoassays for the detection ofanti-gp160 conformational epitope-reactive antibodies. Turning first toimmunoassays, in their most simple and direct sense, preferredimmunoassays of the invention include the various types of enzyme linkedimmunosorbent assays (ELISAs) known to the art. However, it will bereadily appreciated that the utility of the gp160 preparations describedherein are not limited to such assays, and that other useful embodimentsinclude RIAs and other non-enzyme linked antibody binding assays orprocedures.

[0056] In some embodiments of the ELISA assay, native gp160 orappropriate peptides incorporating gp160 antigen sequences areimmobilized onto a selected surface, preferably a surface exhibiting aprotein affinity such as the wells of a polystyrene microtiter plate.After washing to remove incompletely adsorbed material, one will desireto bind or coat a nonspecific protein such as bovine serum albumin(BSA), casein, solutions of milk powder, gelatin, PVP, superblock, orhorse albumin onto the well that is known to be antigenically neutralwith regard to the test antisera. This allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface. Following an appropriate coating period (for example, 3hours), the coated wells will be blocked with a suitable protein, suchas bovine serum albumin (BSA), casein, solutions of milk powder,gelatin, PVP, superblock, or horse albumin, and rinsed several times(e.g., 4 or 5 times) with a suitable buffer, such as PBS. The wells ofthe plates may then be allowed to dry, or may instead be used while theyare still wet.

[0057] After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor from 1 to 4 hours, at temperatures preferably on the order of 20° to25° C. Following incubation, the antisera-contacted surface is washed soas to remove non-immunocomplexed material. A preferred washing procedureincludes washing with a solution such as PBS/Tween, or borate buffer.

[0058] Following formation of specific immunocomplexes between the testsample and the bound antigen, and subsequent washing, the occurrence andeven amount of immunocomplex formation may be determined by subjectingsame to a second antibody having specificity for the first. Of course,in that the test sample will typically be of human origin, the secondantibody will preferably be an antibody having specificity in generalfor human IgG, IgM or IgA. To provide a detecting means, the secondantibody will preferably have an associated enzyme that will generate acolor development upon incubating with an appropriate chromogenicsubstrate. Thus, for example, one will desire to contact and incubatethe antisera-bound surface with a urease, alkaline phosphatase, orperoxidase-conjugated anti-human IgG for a period of time and underconditions which favor the development of immunocomplex formation (e.g.,incubation for 2 hours at room temperature in a PBS-containing solutionsuch as PBS-Tween).

[0059] After incubation with the second enzyme-tagged antibody, andsubsequent to washing to remove unbound material, the amount of label isquantified by incubation with a chromogenic substrate such as urea andbromocresol purple or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonicacid [ABTS] and H₂O₂, in the case of peroxidase as the enzyme label.Quantification is then achieved by measuring the degree of colorgeneration, e.g., using a visible spectra spectrophotometer.

[0060] In each of the microtiter wells will be placed about 10 μl of thetest patient sample along with about 90 μl of reaction buffer (e.g., PBSwith about 1% digitonin or other mild protein solubilizing agent).Control wells of the ELISA plate will include normal sera (human serawithout early anti-HIV antibody), early anti-HIV antibody collected fromHIV patient subjects who had not sero-converted as assessed usingWestern blot, and late anti-HIV antibody obtained from patients thathave seroconverted using conventional anti-HIV antibody detectiontechniques.

EARLY HIV INFECTION DETECTION IN INFANTS

[0061] In a particular embodiment, the invention provides for a methodfor detecting early IgM or IgA anti-HIV antibody in a young child. Thesemethods are improved over existing techniques, as they provide for theearlier detection of HIV infection in infants under the age of 12months.

EARLY ANTI-HIV ANTIBODY DETECTION KITS

[0062] In yet another aspect of the invention, a kit is envisioned forearly anti-HIV antibody detection. In some embodiments, the presentinvention contemplates a diagnostic kit for detecting early anti-HIVantibodies and human immunodeficiency virus infection. The kit comprisesof reagents capable of detecting the early anti-HIV antibodyimmunoreactive with the native or recombinant HIV antigen describedhere.

[0063] In some embodiments, the kit may also comprise a container meanscomprising a secondary antibody capable of detecting the early anti-HIVantibody which is immunoreactive with the recombinant HIV envelopeantigen.

[0064] The HIV antigen reagent of the kit can be provided as a liquidsolution, attached to a solid support or as a dried powder. Preferably,when the reagent is provided in a liquid solution, the liquid solutionis an aqueous solution. Preferably, when the reagent provided isattached to a solid support, the solid support can be chromatographmedia, plastic beads or plates, or a microscope slide. When the reagentprovided is a dry powder, the powder can be reconstituted by theaddition of a suitable solvent. In yet other embodiments, the kit mayfurther comprise a container means comprising an appropriate solvent.

[0065] In some embodiments, the kit comprises a container means thatincludes a volume of a second antibody, such as goat anti-human IgG orIgM conjugated with alkaline phosphatase or other anti-human Igsecondary antibody, and a second container means that includes a volumeof a buffer comprising a non-denaturing solubilizing agent, such asabout 1% digitonin.

[0066] The kit may in other embodiments further comprise a thirdcontainer means that includes an appropriate substrate, such as PNPP foralkaline phosphatase, or 9-dianisidine for peroxidase. A fourthcontainer means that includes an appropriate “stop” buffer, such as 0.5m NaOH, may also be included with various embodiments of the kit.

[0067] The kit may further include an instruction sheet that outlinesthe procedural steps of the assay, and will follow substantially thesame steps as the typical EIA format known to those of skill in the art.

IDIOPATHIC CHRONIC LYMPHOPENIA SCREENING

[0068] In yet another aspect of the invention, methods for screeningsamples for evidence of HIV in idiopathic chronic lymphopenia areprovided. In some embodiments, the method comprises obtaining abiological fluid sample from a patient; exposing said sample to a nativeor recombinant human immunodeficiency virus envelope protein which iscapable of binding early anti-HIV antibodies under conditions sufficientto allow immunocomplex formation between the labeled recombinant proteinand any antibody present in the patient sample, so as to provide anincubation mixture; and identifying the presence of immunocomplexformation in the incubation mixture, via some label, wherein thepresence of labeled immunocomplex formation provides a screen for HIV inidiopathic chronic lymphophenia.

BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0070]FIG. 1. Individuals testing “seronegative” by conventional EIAassay are HIV infected and have detectable early anti-HIV antibody thatimmuno reacts with live cells infected with viral isolates from thepatient. Sera from “seronegative” HIV-infected subjects R6 and R23 weretitrated on live uninfected H9 cells or H9 cells infected with their ownHIV isolates (HIV_(R6) or HIV_(R23)) and stained by FITC-conjugated goatanti-human Ig (polyvalent). The percentage of membrane-fluorescing cellswere scored under a UV microscope, and demonstrates the presence ofearly anti-HIV antibody in the sample.

[0071]FIG. 2. Antibody from “seronegative”, yet HIV-infected subjectreacts with HIV gp160 of HIV-infected cell lysates. SDS-PAGE analysis ofimmunoprecipitates from NP-40 solubilized [³⁵S] methionine-labeled H9cells (uninfected or infected with HIV_(pm213)). Lanes 1-3 and 8 arelysates from H9 infected with HIV_(pm213) and lanes 4-7 are uninfectedH9 lysates. Lanes 1 and 4, R23 serum; lanes 2 and 5, R6 serum, lanes 3and 6, R78 serum; lanes 7 and 8, R58 serum (R23, R6, snf R78 arenegative in routine EIA and Western blot, for HIV antigens, R58 is EIAand WBt).

[0072]FIG. 3. RIP of digitonin-lysed H9 cells infected with HIV₂₁₃.NS=normal human serum; R6 and R23=“early” serum; *=lysate preclearedwith anti-gp160 monoclonal antibody (F105).

[0073]FIG. 4. SDS/PAGE of [³⁵S] methionine-labeled H9 cells infectedwith HIV lysed in different detergents, and immunoprecipitated witheither normal human serum (NS) or serum from patients with earlyanti-HIV antibodies (NIA-0145, 8363).

[0074]FIG. 5. Flow cytometry was used to determine the levels ofanti-gp41 and anti-gp120 mAb bound to three independent clones of HIV₂₁₃gp160-transfected cells (CEM-HIV env-1, env-4, env-7) compared tonon-expressing controls. Cells were stained with 20 μg/ml of control(irrelevant IgG, shaded curve) or anti-g41 mAb (5F3 human mAb, dashedline) or anti-gp120 (gpIII_(2,3) murine mAb, solid line). Detection wasdone using a second species-specific phyco-erytherin (PE)-conjugatedantibody.

[0075]FIG. 6. HIV 213 SalI-Bam I Sequence (nucleic acid sequence)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] The native or recombinant HIV envelope antigen of the presentinvention is substantially undenatured, and has a predominantlypreserved native conformation. Particular HIV strains have beenidentified and used in the description of some exemplary recombinant HIVtarget antigens herein, but are not intended to limit the scope ofdiagnostic/screening assays or products embraced by the claims.

[0077] An EIA that employs recombinant HIV antigens has been developedthat detects the early anti-HIV antibody detected with HIV-infectedlive-cell immunofluorescence. The EIA of the present invention includesa target antigen in which the gp160 HIV protein expressed from vectorsin mammalian cells is solubilized in such a way as to not denature it(such as by use of the solubilizing agent, digitonin), and used to coatwells of an EIA assay plate. Early anti-HIV antibody in patient serareactive with these conformationally preserved, native antigen-mimickingepitopes will be detected using a suitable labeled anti-humanimmunoglobulin (such as alkaline phosphatase-labeled anti-human IgG) or,in the case of infants, anti-human IgM or IgA.

[0078] The following examples are included to demonstrate the existencein HIV-infected subjects who score negative in Western blot or currentEIAs of anti-HIV antibodies that react to conformational epitopes ofgp160. It should be appreciated by those of skill in the art that thetechniques disclosed in the examples which follow represent techniquesdiscovered by the inventor to function well in the practice of theinvention, and thus can be considered to constitute preferred modes forits practice. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

[0079] Several of the methods employed in the following examples aredescribed as follows:

[0080] Methods

[0081] Live-Cell Membrane Immunofluorescence Assays:

[0082] 1. Target cells are acutely-infected H9 or CEM T-cells which areproducing HIV.

[0083] 2. Pipette target cell into centrifuge tubes, and spin 10 minutesat RT. Aspirate fluid, and suspend pellet in media equal to 5× volume ofpellet.

[0084] 3. Add 50 μl of 1° antibody to individual wells of 96-wellround-bottom plates.

[0085] 4. Add 10 μl of target cells to each well.

[0086] 5. Seal and incubate 1 hour at room temperature.

[0087] 6. Spin plate, aspirate fluid, rinse 2 times with media, andaspirate fluid.

[0088] 7. Add 50 μl 2° FITC antibody, appropriately diluted, to eachwell, and incubate 1 hour at room temperature.

[0089] 8. Spin plate (5′×2,000 rpm), aspirate fluid, and rinse cells 2times with media.

[0090] 9. Add 1 drop glycerol/ PBS (1:1) containing 2% formalin to eachwell and mix.

[0091] 10. Mount cell suspension on microscope slides under cover slips.

[0092] Radioimmunoprecipitation and SDS/PAGE Protocols:

[0093] Radiolabeling of Cells:

[0094] 1. For suspension cultures, grow cells to late log phase. Removegrowth medium and wash cells one time with methionine-free culturemedium. Incubate cells for 20-30 minutes at 37° C. to help depletecellular methionine pools.

[0095] 2. Remove wash medium and add a minimal volume of met-freeculture medium containing 50 microcuries/ml of ³⁵S-methionine. Incubatefor 3-4 hours, or as required.

[0096] 3. To harvest cells, collect suspension cultures bycentrifugation and wash one time with cold PBS. Add 1.0 ml of lysingbuffer (0.5% NP40, or 1% digitonin, etc.). Transfer to a microfuge tube,vortex and hold sample at −70° C.

[0097] 4. Determine amount of radiolabeled protein using TCAprecipitation. Just before protein analysis, centrifuge sample for 15minutes in a microfuge to pellet nucleic acids and insoluble debris.

[0098] Immunoprecipitation from Cell Lysates and SDS/PAGE:

[0099] 1. Aliquot an appropriate volume of lysate into a screw-capmicrofuge tube based upon the amount of radiolabeled protein. A typicalRIP might contain 10⁶ cpm. If preclearing is required, add 1:100 (v)antibody (normal serum), vortex and incubate on ice for 30 minutes. Add100 microliters of either Protein A-Sepharose or formalin-fixedStaphylococcus aureus (Cowan strain) to sample, mix and hold on ice for30 minutes. Centrifuge for 5 minutes and recover supernatant andtransfer to new screw-cap tube.*

[0100] Samples (1-2×10⁶ cell equivalents) of precleared lysate areimmunoprecipitated with 3-5 μl of sera or 100 μl of tissue culturemonoclonal antibody supernatant overnight at 4° C. Precipitates arerecovered with S. aureus and analyzed by SDS (10%)-PAGE under reducingconditions (5% 2-mercaptoethanol).

EXAMPLE 1 EARLY ANTI-HIV ANTIBODY WITH LIVE-CELL IMMUNOFLUORESCENCE ASTARGET ANTIGEN

[0101] The present example demonstrates the presence of early anti-HIVantibodies in the sera of human patients previously diagnosed as beingseronegative by conventional anti-HIV antibody detection techniques.

[0102] The presence of early anti-HIV antibodies in 4 of 4 high-riskinfected EIA-, WB-individuals was shown in the present example using anondenaturing test (live-cell membrane immunofluorescence). Theseanti-HIV antibodies were of the IgG isotype, and were found to reactwith their homologous HIV, as well as with a number of other HIV-1isolates (Table 1). TABLE 1 “Seronegative” Patients Possess AntibodyReactive to Multiple HIV-1 Isolates: Summary of Serum Antibody TitersAgainst HIV-Infected Cells Reciprocal of serum dilution that stained 50%of cells H9 infected with Uninfected Serum H9 HIV_(R6) HIV_(R23)HIV_(R58) HIV_(pm213) HIV_(G1) HIV_(AC-1) HIV-III_(B) R6 <10 60 60 10 4020 50 <10 R23 <10 160 160 20 160 640 320 20 R78 <10 30 70 60 15 50 <1040 R82* <10 70 15 30 80 ND 15 ND R58 <10 320 <10 100 1000 200 1000 640

[0103] These sera did not react with HIV-uninfected cells, as controls.

EXAMPLE 2 IMMUNOREACTIVITY OF HIV STRAINS IN CLINICAL SCREENS

[0104] The present example presents a survey of different HIV strains,as the present inventors' earlier studies showed that EIA+, WB+ serafrom HIV-infected individuals possessed a great deal of type-specificityfor native HIV envelope antigens, and are active for different HIVstrains when using live-cell immunofluorescence. Table 2 provides datademonstrating that certain HIV strains appear to be recognized byantibodies from more infected individuals than other HIV isolates. TABLE2 Reactivity of Sera from HIV EIA/WB (+) Individuals for env AntigensExpressed on H9 Cells Infected with Different HIV Isolates HTLV- SERA H9AC-1 CP-1 MCK 0 214 III 205 III_(B) TP-1 AK-1 SK-1 213 G1 Control — — —— 10 — — — — — — — — — Pt. 11 —   20* — — — — 80 20 — — — — 20 —  1 —5120 80 40 — — 40 80 2560 2560 320 20 2560 1280 K —  40 — — 160 160 —5120 160 1280 — 20 2560 —  8 — 5120 20 — 20 — 20 40 5120 2560 160 — 512040 12 —  160 — — 40 — — — — 320 — — — —  5 —  160 80 80 40 — 160 1605120 640 320 — 5120 160  2 — 5120 80 320 160 — 40 1280 160 1280 2560 20640 640 10 — 2560 — 80 160 10 40 40 5120 320 5120 160 5120 80  6 — 2560— — 20 — 320 80 1280 320 — — 1280 — 13 — 5120 — — — 10 80 160 5120 2560160 — 5120 80 15 —  160 — — — — 160 — 640 2560 40 — 5120 40 20 — 5120 20— — — — — 2560 160 2160 160 5120 20 17 — 5120 40 20 80 2560 20 320 1605120 1280 320 1280 640 18 — 2560 — 20 160 5120 — 160 160 1280 320 3202560 160 21 — 1280 — 1280 640 1280 — — 2560 2560 160 320 1280 —  9 —1280 — — — 5120 40 5120 640 — 1280 10 2560 — 14 — 2560 10 20 20 160 201280 640 5120 5120 1280 2560 2560 16 — 1280 — 40 40 2560 160 2560 51201280 1280 80 >640 80

[0105] Other comparisons have been run using “early” sera from infectedhigh-risk individuals (EIA-negative, WB-negative). These data aresummarized in Table 3 which shows that HIV strains 213, AC-1 and C reactwith most of the sera. TABLE 3 Live-Cell Immunofluorescence Reactivityof “Early” Antibodies for Different HIV-1 Strains Total # of Number ofPositive * for These Target Cells Patients H9+ H9+ H9+ H9+ H9+ H9+tested H9 MN RF III_(B) 213 AC-1 C 13 0 0 2 2 11 10 12 (40,80) (40,80)(40-160) (40-80) (40-160)

[0106] Cumulatively, all of the data for both advanced infected as wellas the “early” infected subjects indicate that one or more of thesethree HIV strains will react with antibodies from nearly all, if notall, infected people.

[0107] A larger group of high-risk EIA-negative, WB-negative individualshas also been tested. It was found that antibodies reactive in live-cellmembrane immunofluorescence were present in about 25% of them. Data inTable 4 show the anti-HIV immunofluorescence titers from the singlehigh-risk patient who comprised the study reported in Clerici et al ²⁸.TABLE 4 Membrane Immunofluorescence Titers of Sequentially obtained serasamples from Same Patient Target Cells Serum H9 H9-HIV Visit #8  —  30*Visit #10 — 50 Visit #15 — 70

[0108] This individual possessed low but significant anti-HIV antibodytiters by membrane fluorescence, even though he was “sero-negative” inthe FDA-approved serological tests, and was PCR-negative for HIV²⁸.Correspondingly, he possessed HIV-reactive T-cells, which formed thebasis of their report.

[0109] Table 5 shows the membrane fluorescence titers for another seriesof individuals. The coded samples included a number of normal controls,as well as infected individuals who were sero-positive by the routinetests. TABLE 5 Summary of Membrane Immunoflourescence Reactivity of SeraNumber Positive on These Target Cells Risk Group # of Patients H9H9-HIV₂₁₃ H9-HIV_(AC-1) Heterosexual 10 0  0  0 Low Risk HIV-Infected 100 10 10 EIA/WB-pos. Homosexual  5 0  1  3 High Risk (EIA−, WB−)

[0110] The membrane fluorescence titers corresponded perfectly withthose tests. This group also included five high-risk individuals whowere sequentially bled over a period of eight to ten months. One ofthese individuals (20664) had a low immunofluorescence titer to HIV₂₁₃on the first visit, which was at a time that he was still sero-negativeby the FDA-approved tests. By approximately four months later, thisindividual was positive in EIA and Western blot and had a titer of 1:160by membrane immunofluorescence to HIV₂₁₃, but also to HIV_(AC-1). Thesecond individual (40301) developed a titer by membraneimmunofluorescence to HIV_(AC-1) on his second visit, which increased bythe next visit. A third subject (41593) became sero-positive in themembrane immunofluorescence test on his last visit, but he was negativeby EIA and Western blot. The other two individuals in the group werehigh-risk individuals who never sero-converted by the conventionaltests, and never sero-converted by membrane immunofluorescence.

[0111] Table 6 summarizes the membrane immunofluorescence reactivitiesfor 65 individuals. The groups comprised normal controls, positivecontrols (EIA/Western blot positive), and 36 individuals who werehigh-risk, but sero-negative by EIA and WB. 12 of the latter werepositive by membrane immunofluorescence, and had already been found toalso possess T-cells reactive to HIV peptides. TABLE 6 MembraneImmunofluorescence Titers of Sera No. Membrane Immuno- flourescensePositive No. w/HIV with These Target Cells Risk Group # of PatientsReactive T-Cells H9 H9-213 H9-AC-1 Low Risk 18  0 0  0  0 ControlHIV-Infected 11 ND 0 10 11 EIA+, WB+ High Risk 36 11 0 10 12 EIA−, WB−

[0112] The data from all of the high-risk “seronegative” subjects aresummarized in Table 7. Most of these sera were obtained only once andincludes an additional 50 patient sera. TABLE 7 Summary of Live-CellImmunofluorescence Testing of Sera from High-Risk Subjects forAntibodies to HIV Subject # of Range of Group Subjects # pos TitersHigh-risk 109 29  20-130  Sero-neg. by WB/EIA Low-risk controls  50  0 —AIDS Patients  83 83 100-5000

[0113] 29 of 109 subjects possessed antibodies reactive to live-cellflorescence to at least one of the three HIV strains, eight of thesewere randomly examined for immunoprecipitation of digitonin solubilizedviral protein in infected cells and found that they all reacted with HIVgp160. In live-cell fluorescence, low-risk HIV-NEGATIVE individuals havenot been found positive (i.e., titer≦1:10), and titers in infectedsubjects largely correspond to the stages of disease. High titers inlive-cell fluorescence were usually found in individuals who also scoredpositive in EIA and Western blot, and lower titered individuals usuallydid not score in EIA and Western blot.

RELATIVE SENSITIVITY OF LIVE-CELL IMMUNOFLUORESCENCE ASSAY

[0114] Live-cell immunofluorescence is not necessarily a more sensitiveassay than EIA and WB on all occasions. Control (negative) sera do notstain cells at dilutions greater than 1:10 but do give positivereactions at such low dilutions in EIA and WB. Often control sera needto be diluted to ≈1:100 to be unreactive in EIA and WB, and it is likelythat the high sensitivity of these tests allows scoring of the very lowamounts of antibodies present in human sera which react with mostantigens. About 44-95% of HIV-infected individuals possess significantlevels of auto-antibodies³³. Live-cell fluorescence is too insensitiveto detect these antibodies in sera diluted beyond 1:10. Also, serapositive for anti-HIV antibodies can be diluted out to 1:10,000-1:30,000and remain reactive in EIA and WB, whereas these same sera areunreactive in live-cell fluorescence when diluted beyond 1:5000. Usinglive-infected cells allows detection of antibodies to nativeconformational epitopes, not greater sensitivity of detection ofantibodies on a molar basis.

EXAMPLE 3 SOLUBILIZATION STUDIES—NON-LIVE CELL TARGET ANTIGEN FORDETECTING EARLY ANTI-HIV ANTIBODY

[0115] The present example demonstrates the utility of non-live celltarget antigen for detecting the early anti-HIV antibody in a patientsample. The data here also demonstrates the importance of substantiallypreserved conformational epitopes of the human immunodeficiency virusenvelope protein for detecting the early anti-HIV antibody.

[0116] The effects of different solubilizing agents on HIV proteinimmunoreactivity for early anti-HIV antibody was examined by preparingHIV target antigen with a number of different detergents. This studydemonstrates that early anti-HIV antibodies may be detected in humanserum samples using other than live cells infected with HIV as targetantigen, these samples having previously been determined to beseronegative for antibody using denatured HIV protein as target antigen(in Western blot and conventional EIAs).

[0117] HIV-infected human CEM cells were solubilized with one of theagents listed in Table 8. Each antigen preparation was tested forability to be immunoprecipitated by early anti-HIV antibody contained insera from six patients. These patient sera tested positive for earlyanti-HIV antibody using the live-cell immunofluorescence assay, andnegative for the presence of anti-HIV antibody by conventional WesternBlot assay with denatured HIV target antigen. Each antigen preparationwas also tested with a control serum sample that did not contain earlyanti-HIV antibody, as determined with the live-cell immunofluoresceassay (“NS”=normal sera).

[0118] Table 8 summarizes all the data and shows that digitonin is arepresentative solubilizing agent that may be used to provide the leastdenaturing effect on the protein/peptide for use as a target antigen ascompared to the other detergents examined in this study. These dataprovide evidence that these “early” antibodies recognize conformationalepitopes of HIV gp160. It is contemplated that digitonin atconcentrations of between about 0.1% to about 5% may be used to preparethe target HIV antigen of the present invention without loss of earlyanti-HIV antibody detecting reactivity. TABLE 8 Summary of RIP/SDS-PAGEExamination of Reactivity of “Early” Antibodies to HIV gp160 Solubilizedin Various Detergents “Early” Antibody* Detergent Immunoprecipitation0.5% SDS — 0.5% NP40 3 sera pos; 3 sera neg 10 mM CHAPS — 0.5%Deoxycholate — 1.0% Octylglucoside — 1.0% Digitonin + (all 6) 1 M K₂PO₄— 0.5 mM Dodecylmaltoside — 0.3 mM Theirst —

[0119]FIG. 4 shows representative examples of this work and demonstratesthat “early” antibodies immunoprecipitated gp160 solubilized in about 1%digitonin and 0.5% NP40. Early anti-HIV antibody was detected in allsera samples where gp160 solubilized in digitonin was used as targetantigen. Early anti-HIV antibody was also detected in three out of thesix patient sera samples using the NP40 solubilized HIV target antigens.However, early anti-HIV antibodies were was not detected in any of thesera using HIV antigens treated with 10 mM CHAPS, 0.5% SDS, 0.5%deoxycholate, 1.0% octylglucoside., 1 mM K₂PO₄, 0.5 mM dodecylmaltosideor 0.3 mM Theirst.

[0120] In one embodiment, HIV-infected cells will be solubilized in 1%digitonin, and used to coat EIA plates as the target antigen.

[0121] These radioimmunoprecipitations (RIP) and SDS/PAGE studies alsodemonstrated that the antibodies were reacting to viral antigens, andnot viral-induced cellular antigens. The HIV antigens solubilized withCHAPS did not reveal a detectable immunoprecipitate with early anti-HIVantibody containing sera (FIG. 2, lanes 1 and 2). One of the four seraprovided an immunoprecipitate using 0.5% NP40-solubilized ³⁵S-methioninelabeled infected cells (FIG. 2, lane 4 and 5). Slight denaturation bythis percentage NP40-solubilizing agent destroyed the antigenic epitopesrecognized by the antibodies in the other 3 sera.

[0122] The one reactive sera immuno-precipitated a protein of about160,000 molecular weight, FIG. 2). The protein that wasimmunoprecipitated was confirmed to be HIV gp160 by showing that suchRIP reactivity could be eliminated by precleaning the NP40-cellularlysate with anti-gp160 monoclonal antibody (FIG. 3).

[0123] The following examples outline: (a) development of CEM linesexpressing gp160 from HIV strains AC-1 and C; (b) format of the EIA(i.e., direct coating of the wells with antigen or antibody-capturesandwich assay), including the determination of appropriate blockingreagents to reduce background binding in the EIA; determination studieson the EIA plates in a dry or wet use format, and how to preserve theHIV protein antigenic conformation on the assay plate in commercialembodiment of the invention.

EXAMPLE 4 RECOMBINANT gp160 HIV PROTEINS/PEPTIDES

[0124] The present example demonstrates the utility of the invention forproviding conformationally intact recombinant target antigen that iscapable of immunologically reacting with early anti-HIV antibody. Thepresence of the early anti-HIV antibody is comparable to the anti-HIVantibody detection observed with the live-cell immunofluorescence assay.

[0125] Stable expression of the SalI-XhoI restriction fragment from HIVstrain 213 in a retrovirus vector (pLNSX) in CEM human T-cells hasalready been achieved (FIG. 5). This fragment was blunt-end ligated intothe vector, and following transfection by electroporation, the cellswere selected with G418, and then individual clones were obtained bylimiting dilution and tested for high expression of envelope using F105monoclonal antibody Medarex, Inc. to HIV gp160. The resulting clone(Clone 7) was studied by flow cytometry (FIG. 5) and shown to highlyexpress gp160.

[0126] The Clone 7 was useful in live-cell fluorescence assays and hasbeen observed to react with the early anti-HIV antibodies detected usingH9 HIV infected cells. Cloning of the envelope genes of the other twoHIV strains, HIV_(C) and HIV_(AC-1), has also been accomplished usingthe expression vector system described above.

[0127] Further evidence demonstrating the importance of preservedconformational integrity of HIV target antigen for early anti-HIVantibody binding and detection is provided in the studies conducted bythe inventor using a recombinant gp160 HIV antigen. The envelope ofHIV₂₁₃ was expressed in E. coli. This antigen was then used to attemptimmunoprecipitation of early anti-HIV antibody from patient seraidentified as having early anti-HIV antibody in the live-cellimmunofluorescence assay.

[0128] The recombinant gp160 HIV envelope antigen expressed by E. coliwas not precipitated by the early anti-HIV antibodies in the patientsample, while the digitonin solubilized HIV₂₁₃ infected cell antigendid. These results demonstrated that early anti-HIV antibody is unableto immunologically bind gp160 HIV surface antigen having only apreserved primary structure, as E. coli is not capable of providing arecombinant protein having sufficient conformational characteristicsnecessary for recognition by the early anti-HIV antibody. The overexpression of gp160 in bacteria does not allow for proper folding andglycosylation of the protein.

EXAMPLE 5 DETECTION OF ANTI-HIV ANTIBODY IN INFANTS

[0129] The present examples demonstrates the existence of early anti-HIVantibody in infants, and thus the utility of native recombinant HIVantigen assays for detecting HIV infection in infants.

[0130] Samples examined in these studies were obtained from a pediatricHIV population made up from samples from 48 babies (ages 1 day to 6 mos)born from mothers determined to be HIV sero-positive using conventionalantibody detection techniques. PBMCs were also cultured for HIVdetection by p24 antigen-capture EIA and PCR. HIV was found in six ofthese infants.

[0131] Table 9 summarizes the results of titrations of the infant serain live-cell membrane immunofluorescence against uninfected and infectedcells. TABLE 9 Live-cell immunofluorescence testing of infant sera forIgM reacting with HIV No. Positive for No. Positive for IgM SubjectGroup No. HIV by PCR* to HIV (Titers) to Uninf. Cells Infants born toHIV+ 48 6 6 (10-80) 0 mothers (ages 1 day - 6 mos) Control Infants (ages1 13 0 0 0 day - 5 mos)

[0132] Each of the six infected infants possessed early IgM antibodythat reacted to HIV-infected cells. The sera did not react withuninfected cells, and these sera were also reactive after elimination ofthe IgG by absorption with Sepharose protein G, to eliminate thepossibility that rheumatoid factor was being detected. The uninfectedinfants did not possess anti-HIV IgM, so there was a 100% concordancebetween the presence of IgM to HIV by live-cell fluorescence and thepresence of virus. Noteworthy was the presence of a moderate titer ofanti-HIV IgM in the plasma obtained from a one-day-old baby who wasfound to be infected. The data provides a positive indication that thebaby was obviously infected in utero.

[0133] These data show that infected infants possess HIV-specific IgMwhich can be detected using live-infected cells as antigen. A pediatricHIV-testing kit which will detect IgM and IgA antibody reactive withconformational epitopes of HIV proteins is thus provided for thisparticular application.

[0134] The data supports the contention that HIV-specific T-cells,B-cells, and anti-HIV antibodies which recognize conformational epitopesare produced during the early windows of otherwise immunologicallysilent infection, and eventually infected individuals produce antibodieswhich react to denatured proteins (conventional “sero-conversion” incurrent EIA/WB assays).

[0135] A prospective study by other investigations of infants born toHIV sero-positive mothers shows that out of 12 HIV culture-positiveinfants, 10 had IgA antibody against HIV recombinant protein detectablefrom birth to twenty-seven weeks of age³⁶. In most cases, the first seratested were collected after two months of age. Among children older thanone year, all samples were positive for HIV-specific IgA, while only 50%contained detectable IgM. Notably, only two of thirteen infected infantsunder 3 months of age had IgA detected by their modified Western blotprocedure. Regardless of the testing procedure (Western blot or dotblot) the first serum sample with detectable antibody to HIV wasreactive to envelope glycoprotein gp160.

[0136] The above findings demonstrate the utility of the presentinvention as a new diagnostic test for HIV in infants, that is improvedover current methods as it detects HIV much sooner than standardscreening methods, and is further specific for infection of the infant,and not the mother.

[0137] The present invention in particular embodiments comprises atesting regimen whereby the pediatric subjects may be screened for HIVinfection through early anti-HIV antibody testing via presence ofspecific IgM or IgA to conformational epitopes of HIV gp160, as part ofan improved early diagnosis of pediatric HIV-infection.

EXAMPLE 6 SCREENING METHODS FOR BLOOD IN BLOOD BANKS

[0138] The current tests for HIV infection do not score individuals whohave been recently infected, and even miss a small fraction late in thepre-AIDS period. Since virus may be detected from such EIA-negative,WB-negative patients, they would very likely be able to transmit thevirus to other people during that time. Further, if they donate bloodbelieving that they are not infected, danger to a transfusion recipientof their blood exists. Hence, one of the greatest needs for the presenttypes of test will be extended use in blood bank monitoring of donatedblood. In addition, since many of these types of subjects are mostlikely sexually active, the presently disclosed tests have utility fordetecting early HIV-1 infected individuals who are tested in clinicsdealing with sexually transmitted disease, alternative test sites, etc.

[0139] The present methods provide for the use of recombinant, anon-live cell HIV target antigen that detects the early anti-HIVantibody. The methods in some embodiments comprise an EIA in which gp160expressed from vectors in mammalian cells will be solubilized, such asin digitonin, and used to coat wells of a standard EIA plate. Then,early anti-HIV antibody in patient sera which react with these nativeantigens will be detected using a labeling agent, such as alkalinephosphatase conjugated to anti-human Ig antibody.

EXAMPLE 7 EIA ASSAY WITH NONDENATURED, RECOMBINANT GP160 HIV ANTIGEN

[0140] The present example demonstrates the utility of the invention forproviding an EIA for the detection of early anti-HIV antibodiesemploying a non-denatured, non-live cell HIV target antigen. Thesenon-denatured HIV antigens present a reduced biohazard relative to theHIV infected cell used as target antigen, while providing essentiallythe same anti-HIV early antibody detection activity. The particularnondenatured, non-live cell HIV target antigen illustrated in thepresent example is a recombinant gp160 HIV antigen.

[0141] While the test will provide for the detection of early anti-HIVantibody, it will also demonstrate a positive reaction with lateantibodies detected with conventional anti-HIV antibody detectionmethods.

[0142] The gp160 HIV target antigen will be prepared as described inExample 4 (HIV₂₁₃). However, any HIV envelope protein that may beisolated as part of an encoding nucleic acid sequence and recombinantlyproduced may be used in conjunction with the present invention, saidantigens having the preserved conformational integrity necessary tofacilitate recognition and binding of the early anti-HIV antibody. Inone example, the gp160 expressed from vectors in mammalian cells will besolubilized in about 1% digitonin and used to coat, for example, thewells of a standard 96-well microtiter plate.

[0143] Any early anti-HIV antibody present in a sample, preferably abiological sample (solid tissue or fluid) will immunologically reactwith the antigen, and the early antibody being detected using alkalinephosphatase-labeled anti-human IgG or, in the case of infants,anti-human IgM or IgA.

[0144] The EIA assay may follow any variety of testing formats known tothose of skill in the art, given the information of the presentdisclosure, such as the direct coating of assay wells with antigen orantibody-capture sandwich assay. It is anticipated that the assay platesthat contain or are treated with the antigen may be dried withoutsignificant loss of capacity for binding or attracting the binding ofearly-anti-HIV antibody.

[0145] Determination of EIA Format

[0146] HIV gp160-expressing cells which react to all “early,” as well as“late,” antibodies will be solubilized in a mild solubilizing agent,such as digitonin. This lysate will be size-fractionated on SephadexG2000 columns in the cold, and the peak containing HIV gp160 (asdetermined by RIP) will be used to coat polystyrene microtiter wells at500 ng/well. Following 3 hrs of coating, the wells will be “blocked”with BSA, and rinsed 5 times with buffer. The wells will be eitherallowed to dry or left wet in PBS. 10 μl of “early” antibody will beadded to individual wells containing 90 μl of reaction buffer (PBScontaining about 1% digitonin). The following steps will be typical EIAformat involving incubation for about 1 hour at room temperature, 3rinses with about 1% digitonin-buffer, and incubation with goatanti-human IgG or IgM conjugated with alkaline phosphatase for about 1hour at room temperature (20° C.). The wells will then be rinsed twiceand the substrate added in substrate buffer. Following addition of stopbuffer, the plate will be read on an ELISA plate reader. Controls willinclude wells with no antibody, normal sera, and HIV antibody fromEIA/WB-positive sera.

[0147] If the early antibody significantly registers above normalcontrol sera (i.e., at 4× or greater dilution) in this test, then thisparticular format will be employed in commercial embodiments. It isenvisioned that further refinement may be made in the assay. Inaddition, more highly purified gp160 preparations will be prepared byaffinity purification techniques well known to those of skill in theart, and used as target antigen in the detection of early anti-HIVantibody according to the present invention.

[0148] In the case that digitonin-solubilized material does not stickadequately to plastic plates, several other methods may be used. Onemethod is to first coat the plates with an anti-gp41 or anti-gp160antibody, which would then immobilize gp160 to the plastic in thepresence of digitonin. This sandwich EIA also purifies the antigenduring coating. Several other monoclonal antibodies may also be used toprovide a first coating of antibody to the plates, such as e.g.,monoclonal antibody to gp160, F105, or monoclonal antibody to gp41,chessie 8.

[0149] The present example describes the techniques and agents used toreduce background binding in the EIA of the present invention. A commonproblem with ElAs in the detection of human antibodies is the relativelyhigh degree of background binding with normal control sera. Typically,normal sera needs to be diluted beyond 1:100 in order to be negative inthe test as described here for HIV antibody. The following agents willbe tested as blocking agents, either along or in combination:

[0150] Bovine serum albumin

[0151] PVP

[0152] Superblock

[0153] If high binding with control sera is still observed, otherproteins which will not likely be seen by the human immune system, suchas horse albumin, will be used. The blocking agent(s) that provide thelowest background binding will be selected and used in variousembodiments of the method. Superblock is a commercially availableblocking agent (Pierce Chemical) employed for blocking EIA plates.

[0154] Drying

[0155] The plates will first be dried to assess maintained reactivitywith the early antibodies. Plates may alternatively be treated with asucrose solution before drying to further enhance preservation ofantigen conformational integrity. This technique has been used withother EIAs in order to maintain partial hydration of proteins.Alternatively, glycerin or gelatin may be used, which may preserve theantigen while partially dry, and then upon rehydration can be dissolvedaway as the first step in performing the assay. As yet anotheralternative, the plate wells may be kept wet and the plates well sealedto prevent leakage as a ready to use EIA tool.

[0156] The EIA will be tested against a panel of frozen, banked serathat were collected over a number of years from HIV-infectedindividuals, both sero-positive in the currently employed tests andearly infected individuals who did not score in the current tests.Uninfected control sera will also be examined as controls. All of thesewill be tested undiluted or at various dilutions in the new EIA in orderto determine optimal conditions for testing serum.

EXAMPLE 8 DEVELOPMENT OF OTHER GP160 EXPRESSING CELL LINES

[0157] The present example outlines the method that will be used in thedevelopment of cell lines, such as CEM cell lines, that expressrecombinant HIV antigens. These cell lines will have early anti-HIVantibody reactivity. Exemplary recombinant HIV antigens will includethose of the HIVstrain 213, strain AC-1 and strain C. Cell linesinfected with these recombinant antigens will express an antigen withearly anti-HIV antibody detection activity similar to the HIV-infectedcell lines developed and described here.

[0158] Restriction fragments from a human immunodeficiency virus may beobtained employing known restriction sites that flank nucleic acidsequence encoding the virus envelope protein, or an antigenically activefragment thereof. By way of example, the SalI-XhoI fragments from theparticular HIV isolate, such as the HIV_(AC-1) and HIV_(C) describedhere, may be cloned in a suitable plasmid, such as the pUC19 plasmid.Restriction mapping may be used to confirm that the fragment encodes anHIV envelope protein according to the techniques well known to those ofskill in the art. The obtained fragment may then be ligated into thecloning site of an appropriate vector, such as the pLNSX retrovirusvector (see FIG. 5). The retroviral vector will then be used totransfect an appropriate mammalian cell line, such as mink MiLu cells(ATCC CCL64) or any other line deemed appropriate. The cells may then beselected with a desired selection mechanism, such as antibioticresistance, (e.g. 400-800 micrograms per ml of G418 for 1-2 weeks).Surviving cells from the selection procedure may then be cloned bylimiting dilution in Terasaki plates. Individual clones may then bescreened for expression of recombinant HIV gp160 by live-cellfluorescence (see Example 1) using monoclonal antibody F105 (Medarex,Anandale, N.J.). Clones that express the highest amount of recombinantantigen, based on fluorescence intensity measurements by flow cytometry,may be expanded and cell banks viably frozen.

[0159] The recombinant HIV antigens here described will be useful in thedetection of early anti-HIV antibody, the antigen having conformationalepitopes of the HIV antigen, and particularly HIV envelope antigen suchas gp160, that are maintained and react with early anti-HIV antibodies.Non-live cell preparations of the HIV gp160 antigen are shown tomaintain early anti-HIV antibody immunoreactivity using whole live cellsinfected with recombinant retroviral shuttle vectors carrying the gp160gene fragment solubilized in digitonin. In some embodiments, whole cellsinfected with a retroviral vector carrying a nucleic acid fragmentencoding gp160 will be solubilized in 1% digitonin and used to coat EIAplates. The gp160 expression product from HIV infected cell isolates,such as the HIV₂₁₃, may also be used in the described methods assay.

EXAMPLE 10 COMPARATIVE TESTS OF RECOMBINANT HIV PROTEIN EXPRESSING CELLSAS ANTIGENS VS CELLS INFECTED WITH LIVE HIV BASED ASSAYS FOR ANTIBODY

[0160] For this study, sera from 41 longitudinally-studied high-riskindividuals have been collected. Each serum was tested with therecombinant HIV antigen-expressing CEM cells in live cellimmunofluorescence, as well as three commercially available EIAs forantibodies to HIV: Abbott Laboratories, Electronucleonics, andOrganon-Technica. The assay procedures were those recommended by themanufacturers. Individuals who are found to be positive using the CEMlines, but negative in the three commercial assays, were identified.

[0161] PBLs samples obtained from each of these patients taken at thetime of serum collection, will be analyzed by PCR for HIV DNA, as wellas placed into culture and co-cultured with purified CD4 lymphocytesfrom normal donors. The latter is a very sensitive way of isolating HIVbecause it amplifies it. Therefore, subjects who are only positive inour test will have a confirmatory test of directly detecting HIV in thelymphocytes at the time of serum collection.

[0162] Data from these 41 subjects demonstrate that use of recombinantantigen expressing cells function to detect early anti-HIV antibodysimilar to HIV infected cells. TABLE 10 Assesment of the Presence of“Early” Anti-HIV Antibodies by Live-Cell Immunofluorescence Using CEMCells Expressing Recombinant gp160 Serum CEM CEM-213env CEM-AC1envCEM-Cenv Controls (1) A —* — — — B — — — — C — — — — D — — — — E — — — —F — — — — HIV- infected EIA+/WB+ HR-39 — 1200,640 1280,2500 640,640HR-49 — 1280,1280 1280,2400 640,1000 HR-51 — 320,320 640,640 100,120HR-53 — 320,320 640,640 160,320 R-410 — 640,1000 1280,1280 640,640 HIV-infected High-risk EIA−/WB (2) TL-1 — 60,80 100,80 40,40 HR-2 — 40,5020,40 30,20 HR-9 — 60,80 20,20 20,20 HR-22 — 40,40 60,80 10,20 HR-48 —50,40 —,— 10,10 HR-55 — 100,120 20,40 20,20 HR-56 — 80,100 40,50 40,40

EXAMPLE 11 LIVE- OR FIXED-CELL EIA'S

[0163] The present example demonstrates a particularly envisionedembodiment of the assay plates of the present invention that employ liverecombinant cells that express the desired HIV envelope antigen as atarget antigen. These recombinant live cells may be adhered to the wellsof the plate through a variety of different mechanisms, such as by useof cell lines that must attach to a substrate to grow.

[0164] Particular embodiments of these plates include live- orfixed-cell EIAs in which the wells are plated with live cells expressingthe envelope proteins of HIV strains 213, AC-1, or C. These live cellswould attach to plastic plates. Cell lines that grow by adhering to asubstrate include mink lung fibroblasts (MiLu) as well as otherfibroblastic or epithelial cell lines from human or various animalspecies. The cells would be infected with amphotropic MuLV packagedretrovirus containing the envelope genes of one or any combination ofthe virus strains noted above. The cells would then be selected for G418resistance. The selected cells will then be individually cloned and theclones expressing the highest levels of envelope proteins as assessed bylive-cell immunofluorescence, will be used in the EIA. For the EIA, thecells will be plated into 96-well flat bottom microtiter plates andallowed to attach and grow to confluency.

[0165] The plate can be used with the live cells, or alternatively, thecells can be fixed with a mild fixative (e.g., 0.5% glutaraldehyde) thatwould not destroy the conformational epitopes of gp160 necessary fordetecting early anti-HIV antibodies. Dilutions of test sera as well asknown positive and negative control sera, will be added to individualwells, allowed to incubate for 1-2 hr., and then removed. The wells willthen be washed 3-5× with PBS. A secondary antisera conjugated with anenzyme (e.g., alkaline phosphase) will then be incubated in each wellfor 1-2 hr., followed by three rinses with PBS. Substrate will then beadded to the wells until a visible color is detected. The plate willthen read on an EIA plate reader employing techniques well known tothose of ordinary skill in the art.

[0166] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe composition, methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

[0167] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

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1 1 1 2696 DNA Homo sapiens 1 gtcgacatag cagaataggc gttactcgacagaggagagc aagaaatgga gccagtagat 60 cctagactag agccctggaa gcatccaggaagtcagccta aaactgcttg taccaattgc 120 tattgtaaaa agtgttgctt tcattgccaagtttgtttca taacaaaagc cttaggtatc 180 tcctatggca ggaagaagcg gagacagcgacgaagagctc atcagaacag tcagactcat 240 caagtttctc tatcaaagca gtaagtagtacatgtaacgc aacctatacc aatagtaaca 300 atagtagcct tagtagcagc aataataatagcaatagttg tgtggtccat agtaatcata 360 gaatatagga aaatattaag acaaagaaaaatagacaggt taattgatag actaatagaa 420 agagcagaag acagtggcaa tgagagtgaaggagaaatat cagcacttgt ggagatgggg 480 gtggagatgg ggcaccatgc tccttgggatgttgatgatc tgtagtgcta cagaaaaatt 540 gtgggtcaca gtctattatg gggtacctgtgtggaaggaa gcaaccacca ctctattttg 600 tgcatcagat gctaaagcat atgatacagaggtacataat gtttggacca cacatgcctg 660 tgtacccaca gaccccaacc cacaagaagtagtattggta aatgtgacag aaaattttaa 720 catgtggaaa aatgacatgg tagaacagatgcatgaggat ataatcagtt tatgggatca 780 aagcctaaag ccatgtgtaa aattaaccccactctgtgtt agtttaaagt gcactgattt 840 gaagaatgat actaatacca atagtagtagcgggagaatg ataatggaga aaggagagat 900 aaaaaactgc tctttcaata tcagcacaagcaaaagaggt aaggtgcaga aagaatatgc 960 atttttttat aaacttgata taataccaatagataatgat actaccagct atacgttgac 1020 aagttgtaac acctcagtca ttacacaggcctgtccaaag gtatcctttg agccaattcc 1080 catacattat tgtgccccgg ctggttttgcgattctaaaa tgtaataata agacgttcaa 1140 tggaacagga ccatgtacaa atgtcagcacagtacaatgt acacatggaa ttaggccagt 1200 agtatcaact caactgctgt taaatggcagtctagcagaa gaagaggtag taattagatc 1260 tgtcaatttc acggacaatg ctaaaaccataatagtacag ctgaacacat ctgtagaaat 1320 taattgtaca agacccaaca acaatacaagaaaaaaaatc cgtatccaga ggggaccagg 1380 gagagcattt gttacaatag gaaaaataggaaatatgaga caagcacatt gtaacattag 1440 tagagcaaaa tggaatgcca ctttaaaacagatagctagc aaattaagag aacaatttgg 1500 aaataataaa acaataatct ttaagcaatcctcaggaggg gacccagaaa ttgtaacgca 1560 cagttttaat tgtggagggg aatttttctactgtaattca acacaactgt ttaatagtac 1620 ttggtttaat agtacttgga gtactgaagggtcaaataac actgaaggaa gtgacacaat 1680 cacactccca tgcagaataa aacaatttataaacatgtgg caggaagtag gaaaagcaat 1740 gtatgcccct cccatcagtg gacaaattagatgttcatca aatattacag ggctgctatt 1800 aacaagagat ggtggtaata gcaacaatgagtccgagatc ttcagacctg gaggaggaga 1860 tatgagggac aattggagaa gtgaattatataaatataaa gtagtaaaaa ttgaaccatt 1920 aggagtagca cccaccaagg caaagagaagagtggtgcag agagaaaaaa gagcagtggg 1980 aataggagct ttgttccttg ggttcttgggagcagcagga agcactatgg gctgcacgtc 2040 aatgacgctg acggtacagg ccagacaattattgtctggt atagtgcagc agcagaacaa 2100 tttgctgagg gctattgagg cgcaacagcatctgttgcaa ctcacagtct ggggcatcaa 2160 gcagctccag gcaagaatcc tggctgtggaaagataccta aaggatcaac agctcctggg 2220 gatttggggt tgctctggaa aactcatttgcaccactgct gtgccttgga atgctagttg 2280 gagtaataaa tctctggaac agatttggaataacatgacc tggatggagt gggacagaga 2340 aattaacaat tacacaagct taatacactccttaattgaa gaatcgcaaa accagcaaga 2400 aaagaatgaa caagaattat tggaattagataaatgggca agtttgtgga attggtttaa 2460 cataacaaat tggctgtggt atataaaattattcataatg atagtaggag gcttggtagg 2520 tttaagaata gtttttgctg tactttctgtagtgaataga gttaggcagg gatattcacc 2580 attatcgttt cagacccacc tcccaaccccgaggggaccc gacaggcccg aaggaataga 2640 agaagaaggt ggagagagag acagagacagatccattcga ttagtgaacg gatcct 2696

What is claimed is:
 1. A recombinant protein comprising a recombinanthuman immunodeficiency virus envelope protein capable of immunologicallybinding an early anti-HIV antibody, said protein further defined ashaving a molecular weight of about 160,000 Daltons as determined bySDS/PAGE.
 2. The recombinant protein of claim 1 further defined asprepared by a process comprising: preparing a restriction fragmentcomprising human immunodeficiency virus nucleic acid encoding anenvelope protein gp160; inserting said restriction fragment into avector capable of expressing in a eukaryotic cell; transfecting aeukaryotic cell with said recombinant vector; and collecting arecombinant protein capable of immunologically binding an early anti-HIVantibody.
 3. The recombinant protein of claim 2 wherein the eukaryoticcells in a mammalian cell line, such as CEM or Mu-1-Lu.
 4. Therecombinant protein of claim 3 wherein the eukaryotic mammalian cell isCEM or Mu-1-Lu.
 5. The recombinant protein of claim 2 wherein therecombinant vector is pLNSX-env.
 6. A recombinant vector comprising anucleic acid molecule having a sequence encoding a humanimmunodeficiency virus gp160 protein capable of binding an earlyanti-human immunodeficiency virus antibody.
 7. A mammalian cellcomprising the recombinant vector of claim 6, said recombinant vectorincluding a sequence encoding a gp160 HIV protein.
 8. The mammalian cellof claim 7, wherein the sequence encoding a gp160 HIV protein is aSalI-XhoI restriction fragment from HIV₂₁₃, HIV_(AC-1), HIV_(C), or acombination thereof.
 9. The mammalian cell of claim 7 further defined asa CEM or Mu-1-Lu cell.
 10. The mammalian cell of claim 87, wherein thesequence encoding a gp160 HIV protein is an SalI-XhoI restrictionfragment from HIV₂₁₃ (ATCC VR2247).
 11. A screening method for detectingearly infant infection to human immunodeficiency virus comprising:exposing a biological sample obtained from an infant to a nondenaturedhuman immunodeficiency virus antigen to form an incubation mixture;monitoring the incubation mixture for the presence of immunocomplexformation with the human immunodeficiency virus; and identifying thepresence of IgA or IgM anti-HIV antibody immunobinding to therecombinant antibody, wherein the presence of nondenatured humanimmunodeficiency virus antigen-IgM or IgA antibody immunocomplexformation indicates infection by human immunodeficiency virus in theinfant.
 12. The method of claim 11 wherein the human immunodeficiencyvirus antigen is a recombinant human immunodeficiency virus gp160envelope protein obtained from HIV₂₁₃ (ATCC 2247).
 13. A method forscreening a biological sample for HIV from a patient with IdiopathicChronic Lymphopenia comprising: obtaining a biological sample from apatient having or suspected of having idiopathic chronic lymphophenia;exposing said sample to a nondenatured human immunodeficiency virusantigen under conditions that will allow immunocomplex formation betweenany antibody in the sample and the recombinant protein to provide anincubation mixture; and identifying the presence of immunocomplexformation in the incubation mixture with the labeled humanimmunodeficiency virus antigen by anti-antibody secondary reagents,wherein the presence of immunocomplex formation with antibody in thesample provides a screen for HIV in idiopathic chronic lymphophenia. 14.An assay plate comprising a multiplicity of microtiter wells comprisinga substantially nondenatured and purified human immunodeficiency virusenvelope protein capable of binding an early anti-human immunodeficiencyvirus antibody.
 15. The assay plate of claim 14, wherein the microtiterwells are coated with the substantially nondenatured and purifiedrecombinant human immunodeficiency virus envelope protein.
 16. The assayplate of claim 14, wherein the substantially nondenatured and purifiedrecombinant human immunodeficiency virus envelope protein is furtherdefined as a recombinant gp160 envelope protein.
 17. A kit for screeninga sample for early anti-human immunodeficiency virus comprising: anassay plate as defined in claim 14; a container means comprising labeledsecondary antibody having binding affinity for anti-HIV early antibody.18. A target antigen preparation capable of immunologically binding anearly anti-HIV antibody comprising a recombinant HIV envelope proteingp160 obtained from a purified eukaryotic cell lysate, said eukaryoticcell lysate comprising a lysate from HIV infected mammalian cells, saidcells being infected with HIV_(AC-1), a mammalian cell infected withHIV_(C) and a mammalian cell infected with HIV₂₁₃.
 19. The targetantigen of claim 18 wherein the mammalian cell lysate is prepared by aprocess of solubilizing the HIV expressing mammalian cells with about0.1% to an about 10% digitonin.
 20. The target antigen of claim 18,wherein the mammalian cells are CEM cells.
 21. The target antigen ofclaim 18, wherein the mammalian cells are Mu-1-Lu cells.
 22. An assayplate for use in detecting early anti-HIV antibody comprising cellsadherent to the assay plate expressing recombinant gp160 from a viralisolate selected from the group consisting of HIV₂₁₃, HIV-_(AC-1), andHIV_(C).
 23. The assay plate of claim 22 wherein the cells are Mu-1-Lucells.